Metastatic melanoma is the most aggressive form of skin cancer and a devastating disease. The development of immune checkpoint inhibitors (ICI) represents a major therapeutic improvement in melanoma care, inducing durable responses in a portion of patients. However, the majority of patients has intrinsic or acquired resistance to ICI and derives no benefit from these therapies. The molecular underpinnings of ICI-resistance are poorly understood. The overarching goal of this proposal is to determine mechanisms of ICI-resistance in melanoma using several experimental and technological innovations. We performed single-cell RNA- sequencing (sc-RNA-seq) and multiplexed imaging of ICI-resistant melanoma tumors, and a genome-scale gain-of-function screen in patient-derived melanoma cell lines and their corresponding tumor infiltrating lymphocytes (TILs). Our sc-RNA-seq analysis revealed a strong association between melanoma cell- autonomous expression of angiogenesis pathways with ICI-resistance. In line with this finding, our functional genetic screen identified KDR (also known as VEGFR2) as top hit of putative mediators of immune escape. KDR is a major receptor for vascular endothelial growth factor (VEGF) and induces the expression of angiogenesis pathways that were identified by sc-RNA-seq. Multiplexed imaging confirmed protein expression of KDR at the invasive tumor front, a key site of tumor-immune interactions in melanoma that predicts response and resistance to ICI. Together, these preliminary results highlight the putative role of KDR and downstream angiogenesis pathway expression in ICI-resistance. This proposal builds on these findings with the specific focus to: 1) dissect the mechanisms of KDR-mediated immune escape, 2) validate transcriptional and protein expression of KDR and its downstream angiogenesis pathways in patients undergoing serial biopsies while receiving ICI therapy, and 3) functionally validate mechanisms of ICI-resistance in isogenic patient-derived cell lines. Results of these studies have the potential to guide novel drug combination strategies that could be rapidly translated into clinical application. Dr. Benjamin Izar is mentored by Dr. Kai Wucherpfennig, a physician-scientist and expert in immuno-oncology, and Dr. Aviv Regev, a pioneer in sc- RNA-seq and computational analyses. Dr. Izar has a committed advisory committee comprised of Drs. Stephen Hodi, Keith Flaherty and Peter Sorger, who will provide additional mentorship and collaboration in immuno-oncology, cancer biology and pharmacology. Dr. Izar has developed a 5-year training plan with a detailed outline of activities that will facilitate his development to an independent investigator. Dr. Izar will leverage an exceptional research environment at Dana-Farber Cancer Institute, Harvard Medical School and the Broad, and a richness of scientific meetings, professional development seminars and didactic coursework.